Optimizing Enzyme-Linked Immunosorbent Assay Parameters for Chlamydial Abortion Diagnosis in Sheep

Kogarshyn Matkerimova, Khairulla Abeuov, Kydyrbay Maikhin, Abdikalyk Abishov, Maxat Berdikulov, Assiya Mussayeva, Bolat Yespembetov, Yessengali Ussenbekov, Berikzhan Kayypbai and Nazym Syrym

Background and Objective: Chlamydial abortion is one of the leading causes of reproductive loss in sheep, yet diagnostic accuracy remains limited due to suboptimal assay conditions. To address this gap, the present study aimed to optimize the conditions of an Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of chlamydial abortion in sheep, using TT and MM strains of Chlamydia previously isolated from affected animals. Materials and Methods: Yolk sac suspensions from 6-7-day-old chicken embryos infected with the causative agent of sheep enzootic abortion were used as starting material for antigen preparation. The MM strain was employed to develop an optimal method for the purification and concentration of antigens. Hyperimmunization schemes were designed using purified proteins in combination with biostimulants to obtain positive serum. Conjugates were prepared from isolated immunoglobulins, achieving an activity of 1:800. Immunoglobulins were purified by triple precipitation with saturated ammonium sulfate solution, followed by gel chromatography on Sephadex G-200 and identification by immunoelectrophoresis. Statistical analyses were performed using Student’s t-test (p≤0.05) for continuous data and a one-sided Fisher’s exact test (α<0.05) for comparing group efficacy. Results: The study established optimal contact parameters for antigen antibody interactions in the ELISA system. Results showed that effective binding occurred within 3 hrs at 37±1°C or 18 hrs at 4±2°C, while specific conjugate-antigen interaction was optimal for 1 hr at 37±1°C. These optimized parameters significantly enhanced assay sensitivity and reliability, providing reproducible detection of antibodies against the chlamydial abortion pathogen. Conclusion: This study developed an optimized ELISA protocol for the diagnosis of sheep enzootic abortion by refining antigen preparation, immunoglobulin purification and incubation conditions. The findings contribute to improving diagnostic accuracy and may serve as a foundation for developing more rapid and cost-effective diagnostic kits for field application. Future research should validate these optimized conditions across broader sheep populations and different chlamydial strains.

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